Fluorometric Measurement of Calmodulin-Dependent Peptide–Protein Interactions Using Dansylated Calmodulin

The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions. Key features • Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding. • Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts. • Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development. • Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.81961 The assessment of peptide-protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides.In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring.In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan.Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes.Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance.DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time.In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein.This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions.

Background
Calmodulin (CaM), a pivotal Ca 2+ -binding protein, intricately regulates essential biological functions by binding to the cation, thereby exerting meticulous control over an array of effector proteins.This interaction induces conformational changes in CaM, critically influencing cellular processes such as muscle contraction and neurotransmitter release, as exhaustively elucidated by Chin and Means [1] and Rhoads and Friedberg [2].The trajectory of unraveling CaM's multifaceted role spans decades, commencing in the 1970s with the identification of cyclic nucleotide phosphodiesterase as one of the initial proteins binding to CaM, as underscored by Rasmussen et al. [3].Subsequently, Klee and Vanaman's seminal work in 1982 laid the foundational understanding of CaM's centrality in cellular signal transduction.The ongoing delineation of over 300 target peptides for CaM, meticulously documented by Klee and Vanaman [4], accentuates its indispensability in diverse cellular processes.Technological advancements, such as dansylation [5] and fluorogenesis [6], have significantly contributed to the precise identification and characterization of CaM targets, thereby enhancing our understanding of fundamental cellular processes.Among these tools, dansyl-CaM (D-CaM), a derivative of CaM conjugated with dansyl chloride (DNSC) [5-(dimethylamino)naphthalene-1-sulfonyl chloride], emerges as a distinctive and powerful instrument for analyzing interactions with peptides and proteins.DNSC specifically interacts with primary amino groups, forming stable blue or blue-green fluorescent sulfonamide adducts with aliphatic and aromatic amines (Tyr, Phe, Trp, etc.).The incorporation of dansyl into CaM enables the sensitive detection and thorough examination of this modified protein using fluorescence-based techniques.High-resolution structural scrutiny of apo-CaM and holo-CaM has unveiled intricate Ca 2+ -induced structural changes, laying bare hydrophobic interfaces, aligning with the observations of Chin and Means [1]

A. CaM expression and purification
The human CaM gene, inserted into the pET-14b expression vector, is introduced into BL21-DE3 E. coli (other bacteria strain to express non-toxic heterologous genes can also be used).The purification procedure for CaM has been adapted from existing literature [11] and results in substantial yields of soluble protein, as outlined below.

Protein expression
a. Cultivate BL21-DE3 cells from glycerol stock in 1 L of LB medium at 37 °C, supplemented with 100 μg/mL ampicillin, until the optical density (A600) reaches 0.8-1.b.Induce protein expression by adding 0.4 mM IPTG and continue cultivation for 4-6 h at 37 °C or overnight at 20 °C.

Cell harvesting and resuspension
a. Centrifuge the cells to collect them (9,000× g for 9 min at 4 °C) and wash the cell pellet twice with 50 mL of fresh lysis buffer.b.Resuspend the cell pellet in 30 mL of lysis buffer and store the sample in 10 mL aliquots at -20 °C.

Sample preparation
a. Thaw an aliquot on ice and perform sonication (three cycles of 10 s at 50 kHz; keep the sample on ice).Alternatively, employ the Bradford method for quantification.To determine the specific dansylated residues in CaM, tryptic digestion coupled with mass spectroscopy or gas-phase protein sequencers have been employed, reporting dansylation at either Lys75 or Lys 115 [12,13].The mass spectrometry analysis revealed the binding of up to four dansyl molecules per CaM.Furthermore, tandem mass spectrometry of tryptic peptides strongly indicates dansylation at Ala1 and Lys148 [14].The identification of the remaining two dansylated residues is pending further investigation; however, the data are consistent with the possibility of them being Lys75 and Lys115 [14].

Fraction analysis
Swiftly verify the presence of the protein in the collected fractions by performing dot blotting on nitrocellulose and staining it with Ponceau Red.Additionally, analyze the fractions using 15% SDS-PAGE gels.For a more detailed examination, record the emission spectra of each sample (as further explained This process allows for the efficient dansylation of CaM, making it ready for various downstream applications and analyses.

C. Peptide resuspension
When working with peptides, start by dissolving them in distilled, sterile water.This is especially suitable for short peptides (<5 residues).For each specific peptide, choose the most appropriate conditions to ensure optimum solubility based on its sequence.
1. Calculate overall charge; begin by assessing the overall charge of the peptide: Assign a value of -1 for each acidic residue, including aspartic acid (Asp or D), glutamic acid (Glu or E), and the C-terminal -COOH.Assign a value of +1 for each basic residue, including arginine (Arg or R), lysine (Lys or K), histidine (His or H), and the N-terminal -NH2.Calculate the net charge of the peptide.a. Positive charge peptides; if the overall charge of the peptide is positive: Attempt to dissolve the peptide in water initially.If water does not work, try a 10%-30% acetic acid solution.If the peptide still does not dissolve, add a small amount of TFA (<50 μL) to solubilize it and then dilute to the desired concentration.b.Negative charge peptides; if the overall charge of the peptide is negative: Start by attempting to dissolve the peptide in water.If water is ineffective to dissolve the peptide, add a small amount of NH4OH (<50 μL) and dilute to the desired concentration.See Note 9. c.Neutral charge peptides; for peptides with a net charge of zero: Introduce organic solvents as follows: First, try adding acetonitrile, methanol, or isopropanol.For highly hydrophobic peptides, start with a small amount of DMSO (30-50 μL, 100%).Gradually add this solution drop by drop to a stirring aqueous buffered solution like PBS or your preferred buffer until the desired peptide concentration is achieved.If turbidity appears in the peptide solution, it indicates that you have reached the limit of solubility.In such cases, sonication can help to dissolve the peptides.If the peptide includes cysteine residues, use DMF instead of DMSO.See Note 10.
In cases where peptides tend to aggregate, incorporate 6 M guanidine•HCl or 8 M urea before proceeding with necessary dilutions.

D. Peptide interaction with Apo-CaM
1. Dilute the peptide stock in fluorescence buffer to have a 20 μM peptide solution.
2. Calculate the volumes of peptide required for each titration step using the formula: C1•V1 = C2•V2, where C1 is the initial peptide concentration in the stock (20 µM), V1 is the volume of peptide to add in each step, C2 is the desired final peptide concentration in each step, and V2 is the total sample volume (100 µL).Prepare a table with the calculated volumes (Table 1): 3. Following sample preparation, centrifuge them at 185,494× g (radius 98 mm) for 10 min and carefully transfer the resulting supernatants to fresh 500 µL tubes to remove any potential aggregates.Ensure the absence of air bubbles as they can distort fluorescence readings.Insert the cuvette into the fluorimeter and proceed to acquire emission spectra.Employ an excitation wavelength of 340 nm, recording emissions across the 400-660 nm range.All measurements are conducted at a temperature of 25 °C, with necessary corrections applied to account for any buffer-related interference.These conditions should yield a prominent fluorescence peak around 500 nm in the D-CaM spectrum.Record the fluorescence values for each peptide concentration.4. Analyze data to determine the interaction between the peptide and D-CaM at different concentrations.

Calcium titration:
1. Start the process by gradually adding concentrated CaCl2 aliquots into a cuvette containing the peptide saturation sample once the peptide concentration for signal saturation is achieved.2. In a 100 μL sample of the peptide saturation sample, add 1 μL of Ca buffer solution sequentially.It is important to note that all experiments will be conducted under constant conditions of pH 7.4 and 25 °C.Remember that EGTA buffering is pH dependent.It is worth emphasizing that the experimental design incorporates a 10 mM Ca buffer, affording the evaluation of free calcium concentrations spanning from nM to µM ranges.Notably, adjustments in CaCl2 concentration are permissible, and alternative buffers may be employed to facilitate analyses across diverse calcium concentration ranges, whether elevated or diminished.3. Utilize the Ca-EGTA Calculator v1.3, incorporating constants sourced from Theo Schoenmakers' Chelator, particularly Table 2, which corresponds to the 10 mM CaCl2 calcium buffer.After each Ca 2+ addition, ensure thorough mixing to attain homogeneity.See Note 11.

E. Peptide interaction with Holo-CaM
1. To evaluate the peptide's interaction with holo-D-CaM, first saturate CaM with 1 mM free CaCl2.Calculate the necessary peptide volumes for each titration step using the equation: C1•V1 = C2•V2, where C1 represents the initial peptide concentration in the stock (20 μM), V1 is the volume of peptide to be added in each step, C2 is the desired final peptide concentration for each step, and V2 is the total sample volume (100 μL).Create a table to document the calculated volumes (Table 3): 2. After preparing the samples, centrifuge them at 185,494× g (radius 98 mm) for 10 min and carefully transfer the resulting supernatants to fresh 500 μL tubes to eliminate any potential aggregates.3. Ensure the absence of air bubbles, as they can distort fluorescence measurements.Insert the cuvette into the fluorimeter and proceed to acquire emission spectra.Use an excitation wavelength of 340 nm, recording emissions across the 400-660 nm range.All measurements are conducted at a temperature of 25 °C, with appropriate corrections made to account for any buffer-related interference.These conditions should yield a prominent fluorescence peak around 500 nm in the D-CaM spectrum.Record the fluorescence values for each peptide concentration.

Data analysis
In evaluating the affinity of a specific peptide for D-CaM, our approach involves the analysis of emission spectra obtained at various peptide concentrations, depicted in Figure 1A.Notably, a correlation is observed between peptide concentration and the intensity of the emission spectrum.The emission spectra exhibit a discernible trend until reaching a saturation point, wherein further increments in peptide concentration no longer elicit changes in the maximal emission intensity.
To better assess changes in intensity, we focus on the wavelength range of 490-500 nm (Table 4).We quantify intensity increases by summing up the values within this range.For normalization, we use the apo-CaM condition as the baseline, ensuring that D-CaM with 0 μM peptide corresponds to an intensity of 0. To normalize, we divide the sum of intensities by the corresponding value obtained in the apo-CaM condition (Table 4).The experimental protocol is subsequently reiterated under Holo-CaM conditions, wherein a saturating concentration of unbound Ca 2+ (e.g., 10 mM) is initially introduced, followed by the repetition of the peptide titration process.
For a more lucid depiction of intensity changes, we present the normalized values as percentages, setting the maximum intensity as 100.This normalization entails dividing each value by the maximum intensity obtained and then multiplying the result by 100 (Table 1).This approach facilitates a robust comparison of intensity changes across various peptide concentrations, culminating in a percentage-based representation of the specified peptide's affinity for D-CaM.
To generate concentration-response curves, plot fluorescence enhancement against the peptide-D-CaM ratio or [peptide] and fit the data using the three-parameter Hill equation through curvilinear regression.EC50 values vary with D-CaM concentration due to ligand depletion, especially at low concentrations.Correct for depletion by determining EC50 values across a range of D-CaM concentrations.At infinitely low D-CaM concentrations, depletion should be negligible, making EC50 a true affinity value [1].For accurate dissociation constant (Kd) determination, perform titrations with varying initial concentrations of D-CaM (6.25-200 nM) and create concentration-response curves at each D-CaM concentration (Figure 1B).Calculate apparent dissociation constants (EC50) from Figure 1B and plot them against D-CaM concentrations (Figure 1C).Obtain true dissociation constants through linear fitting and extrapolation to D-CaM concentrations equal to zero.If stoichiometry is known, estimate Kd values using a Scatchard plot analysis.However, we recommend the previous method for its accuracy and reliability in Kd determination, particularly in complex binding interactions.To estimate Kd, assuming a 1:1 stoichiometry, apply the equation: Here, F represents the increase in fluorescence, Fmax is the maximal fluorescence (variable), [peptide] is the known total peptide concentration, [CaM] is the known concentration of total D-CaM, and Kd is the variable for the affinity constant.Express results as means ± S.E.M from three or more experiments.For statistical analysis, use the unpaired Student t-test, with P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) considered statistically significant.This protocol provides a comprehensive method for estimating the Kd in a fluorescence-based ligand displacement assay with D-CaM, considering the impact of D-CaM concentration and correcting for ligand depletion at low concentrations.It offers a robust approach for characterizing the binding affinity and cooperativity between D-CaM and the ligand.

Validation of protocol
This methodology, initially validated by Kincaid and Vaughan in 1986 [17], has exhibited consistent efficacy in discerning conformational changes resulting from interactions with Ca 2+ , peptides, or proteins.Subsequent studies Yuan by and Graves in 1989 [18] investigated the interaction of CaM with the γ subunit of phosphorylase kinase, while Munier et al. in 1991 [19] characterized the catalytic and calmodulin-binding domains of Bordetella pertussis Cite as: Nuñez, E. et al. (2024).Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.Bio-protocol 14(7): e4963.DOI: 10.21769/BioProtoc.4963.9 Published: Apr 05, 2024 b.Subject the sample to three freeze-thaw cycles by alternating between a dry ice ethanol bath and a 37 °C water bath.c.Centrifuge the sample in a microcentrifuge at 14,000× g for 15 min.d.Heat the supernatant to 95 °C for 5 min, followed by centrifugation as previously described.This step leverages CaM's enhanced thermal stability.4. Chromatography a. Introduce CaCl2 to the supernatant (final concentration: 5 mM).Load the sample at room temperature onto a 5 mL Phenyl-Sepharose column pre-equilibrated with CQ buffer.The chromatography can be achieved through gravity flow or by utilizing a peristaltic pump.Wash the column with 20 column volumes of CW buffer followed by 10 column volumes of CHSW buffer.b.Elute CaM with 20 column volumes of CE buffer, taking fractions of 500 µL. 5. Analysis and storage a. Mix 20 µL of CaM with 5 µL of 5× loading buffer.Load the mixture onto a 15% acrylamide gel and perform electrophoresis using a 1× dilution of the running buffer.Run the gel at a voltage approximately 120-150 V for 90 min.Stain the gel with Coomassie Blue for 10 min and destain first using fast destaining for 15 min followed by regular destaining for 30 min.Concentrate the sample to have a final concentration of at least 1 mg/mL using an Amicon centrifugal filter of 3 kDa.Dialyze the fractions against MilliQ water, b.Store the purified CaM at -20 °C at 1 mg/mL in fractions of 1 mL or lyophilize it in fractions of 1 mL.c.CaM concentration estimation can be done via absorbance at 276 nm, with ε276 = 3,030 M -1 •cm -1 .

4 .
Separation of dansylated CaM (D-CaM) a.To separate D-CaM from any unreacted dansyl chloride, you will need a disposable column packed with approximately 1 mL of Sephadex G-25.b.Equilibrate approximately 250 mg of dry resin with distilled water.c.Load the D-CaM mixture onto the column and collect fractions of 50-100 μL each.d.The initial fractions eluted, known as the "excluded fraction," contain the D-CaM conjugate.See Note 7.

10 Published 6 . 7 .
Cite as: Nuñez, E. et al. (2024).Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.Bio-protocol 14(7): e4963.DOI: 10.21769/BioProtoc.4963.D-CaM storage a. Gather the fractions containing D-CaM and concentrate them if needed.We recommend stocks between 0.5 and 5 µM.b.Store the resulting D-CaM aliquots in a dark environment at -20 °C or, alternatively, as lyophilized samples.Typically, the conjugate retains its properties when stored at -20 °C for several months or more.See Note 8. Protein concentration determination Utilize the Bradford assay to determine the protein concentrations of D-CaM, using unlabeled CaM as a standard.Alternatively, measure the concentration of D-CaM by UV absorption at 320 nm (ε320 = 3,400 M -1 •cm -1 ).Dansyl moiety concentration: Determine the concentration of the incorporated dansyl moiety via spectroscopy.When possible, calculate the number of specific dansylated residues within a D-CaM molecule.

12 Published 4 .
Record the fluorescence spectra approximately 20-30 s after adding each sample to the cuvette.Note that extended equilibration times do not yield improved data. 5. Continue the titration of Ca 2+ until saturation is reached, signified by the absence of further observable changes in the spectra.
1. CaM preparationBegin by diluting CaM in D buffer to achieve a final concentration of 1 mg/mL.2.Dansyl chloride preparationa.Dissolve dansyl chloride in acetone at a concentration of 2.17 mg/mL.b.Store this dansyl chloride solution at either 4 °C or -20 °C in a dark environment.It remains stable for an extended period, often several months.3.Dansylation processa.

Table 4 .
Experimental data summary.The values in the Normalized data column are obtained by dividing each intensity value by the corresponding value obtained in apoD-CaM and subtracting 1.The Percentage column represents the percentages, with the maximum taken as 100%, calculated by dividing all values by the maximum normalized value.If we plot the obtained values against the peptide concentration, a dose-response curve in percentage is generated.